This invention relates to diagnostic and/or therapeutically active agents comprising gas microbubbles, more particularly to such agents comprising lipopeptide-stabilised gas microbubbles. These agents if desired may incorporate moieties having affinity for sites and/or structures within the body so that diagnostic imaging and/or therapy of particular locations within the body may be enhanced. Of particular interest are diagnostic agents for use in ultrasound imaging. Novel lipopeptides constitute a further feature of the invention.
It is well known that ultrasonic imaging comprises a potentially valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography, and of tissue microvasculature. A variety of contrast agents have been proposed to enhance the acoustic images so obtained, including suspensions of solid particles, emulsified liquid droplets, gas bubbles and encapsulated gases or liquids. It is generally accepted that low density contrast agents which are easily compressible are particularly efficient in terms of the acoustic backscatter they generate, and considerable interest has therefore been shown in the preparation of gas-containing and gas-generating systems.
Initial studies involving free gas bubbles generated in vivo by intracardiac injection of physiologically acceptable substances have demonstrated the potential efficiency of such bubbles as contrast agents in echography; such techniques are severely limited in practice, however, by the short lifetime of the free bubbles. Interest has accordingly been shown in methods of stabilising gas bubbles for echocardiography and other ultrasonic studies, for example using emulsifiers, oils, thickeners or sugars, or by entraining or encapsulating the gas or a precursor thereof in a variety of systems, e.g. as porous gas-containing microparticles or as encapsulated gas microbubbles.
There is a substantial body of prior art concerning the nature of encapsulating materials and gases which may be present within microparticles, microbubbles etc. One preferred encapsulating system uses negatively charged phospholidids as wail-forming materials to stabilise gas microbubblesxe2x80x94see WO-A-9729783, which is hereby incorporated by reference and which contains a comprehensive review of prior art in this area. Despite a large amount of research there still remains a need for stabilised gas-filled microbubbles or microparticles which can act as ultrasound contrast agents and which are both physiologically tolerable and echogenic. Many existing contrast agents, for example, are destroyed by continuous ultrasound exposure, and thus any enhancement in contrast agent stability may reduce this problem.
It has recently been found that certain peptides with alternating hydrophobic and hydrophilic residues may spontaneously form macroscopic peptide membranes which may be useful biomaterials for medical products, for example as vehicles for slow-diffusion drug delivery, separation materials, biodegradable polymers and artificial sutures. U.S. Pat. No. 5,670,483 describes membranes formed by the peptide EAK16 derived from the protein zuotin [see also Zhang, S in Biopolymers (1994) 34, 663; Zhang, S in Biomaterals (1995) 16, 1385; and Zhang, S in Proc. Natl. Acad. Sci (1993) 90, 3334]. The membranes are stable in aqueous solutions and are resistant to degredation by heat, enzymatic degradation and alkaline and acidic pH; they have also been found to be non-cytotoxic. These peptides are soluble in aqueous solutions and, according to U.S. Pat. No. 5,670,483, require a sequence length of at least 12 amino acid residues, preferably more than 16 residues, in order to form membrane structures.
Fujita, K. et al. in Advances in Biophysics (1997) 34, 127 have described supramolecular assemblies using helical peptides. When such peptides were suspended in an aqueous medium by a sonication method, a dispersion of vesicles termed xe2x80x9cpeptosomesxe2x80x9d was obtained. These peptosomes had a similar size distribution to classical liposomes, i.e. in the nanometer range; typically their average particle size was 75 nm. Other molecular assemblies comprising peptidic structures have been described by Imanishi, Y. et al. in Supramol. Sci (1996) 13, where gramicidin A/PEG conjugates were found to form peptosomes also in the nanometer size range.
It has now been found that a range of lipid-substituted peptide derivatives, referred to herein as lipopeptides, may be used in the formation of stabilised gas microbubbles suitable for use as diagnostic and/or therapeutic agents, for example in ultrasound echography. Such microbubbles have been found to exhibit good stability, for example during ultrasonication in an imaging procedure. It has also surprisingly been found that lipopetides containing as few as two amino acid residues may exhibit membrane forming properties, in contrast to the findings regarding the self-assembly peptide structures of U.S. Pat. No. 5,670,483. Such short lipopeptides may be prepared relatively easily and economically and may therefore possess substantial cost advantages over naturally occurring, semi-synthetic or synthetic phospholipids such as phosphatidylserine.
Thus according to one aspect of the present invention there is provided a diagnostic and/or therapeutically active agent, e.g. an ultrasound contrast agent, comprising encapsualted gas-filled microbubbles stabilised by membrane-forming amphiphilic lipopeptides.
Viewed from another aspect the invention provides the use of an agent as hereinbefore defined as an ultrasound contrast agent.
Viewed from yet another aspect, the invention provides a method of generating enhanced images of a human or non-human animal body which comprises administering to said body an agent as as hereinbefore defined and generating an ultrasound, magnetic resonance, X-ray, radiographic or light image of at least a part of said body.
The macroscopic membranes may be formed from individual peptide units comprising from 2 to 50 aminoacyl residues. Each peptide unit may carry one or more lipophilic hydrocarbon chains of between 5 and about 50 carbons in length.
In a preferred embodiment the number of amino acid residues in the individual lipopetide units of the invention should be the least number of residues necessary to form an effective stabilised membrane and is preferably less than 20 residues, more preferably less than 10 residues, most preferably between 2 to 8 residues. Clearly, keeping the number of residues to a minimum will both reduce cost and allow easier preparation of the lipopeptides of the invention.
Any amino acid residue may be used in the preparation of individual lipopetide units according to the invention, although the lipopeptides must be amphiphilic. In a preferred embodiment the lipopeptides will comprise residues of amino acids selected from the readily available naturally occuring essential twenty amino acids.
In one embodiment a peptide unit can comprise alternating hydrophobic and hydrophilic residues, such as alanyl and diaminopropionyl, and may comprise one or more complementary sequences and/or a targeting sequence with affinity for biological receptors. In a particularly preferred embodiment, charged amino acids such as lysine and glutamic acid are selected to provide side-chain functionalities comprising positively and/or negatively charged groups respectively at neutral pH. Although not wishing to be limited by theory, it is envisaged that these charged groups help in the stabilisation of the-outer part of the membrane by forming ion-pairs or salt bridges. The alignment of oppositely charged groups leading to membrane stability is possible only if the peptide sequences involved are complementary to one another and this forms a further aspect of the invention.
The lipid component of the lipopeptides preferably comprises an alkyl, alkenyl or alkynyl chain, especially an alkyl chain. The hydrocarbon chains preferably have between 5 and 25 carbons and most preferably are obtainable from readily available fatty acid derivatives. Suitable fatty acids include oleic acid, stearic acid, palmitic acid and the like; such fatty acids are well-known to the person skilled in the art. The number of hydrocarbon chains per individual lipopetide unit will vary depending on the number of amino acid residues present and will be readily determined by the person skilled in the art; typically each lipopeptide molecule will comprise one or two hydrocarbon chains.
The peptide chains may comprise amino acid sequences that will attain self-stabilising secondary structures such as beta sheets or alpha helices. These may provide the membranes and corresponding microbubbles with advantageous perfomance characteristics such as stability, pharmacokinetics, biotolerability or receptor affinities. A beta sheet-forming lipopeptide, for example such as palmitoyl-(Glu-Ile-Lys-Ile)2, will be stabilised by repeat of counterion and hydrophobicity, and may provide the surface with both ionic and hydrophobic stabilisation.
In addition to the amino acid sequences of the lipopeptides themselves having a stabilising effect, fatty acyl chains linked to amino acid residues in the lipopeptides may be selected to provide the structure with certain characteristics. Thus, for example, mixtures of cis- and trans-unsaturated acyl chains will add to the amorphous nature of the membranes, thereby allowing greater membrane flexibility, especially at higher ultrasound frequencies, e.g. providing better second harmonic signals. A similar increase in amorphous nature or reduction in crystallinity of lipid structures may be obtained by incorporating branched fatty acyl chains, including mixtures of acyl chains with differently located branching.
Alpha helices may be formed in lipopeptides for certain amino acid sequences, as is well known in the art of protein chemistry. In such sequences a number of hydrogen bonds between side chains of properly separated and selected amino acids will serve to keep the peptide chain in alpha helix structures. For example, a structure such as Lys-Lys(acyl)-Gln-Lys(diacyl)-Asn-Lys(acyl)-Gln-Leu will provide strong hydrogen bonding between the Asn and Gln side chains and provide a polar, uncharged surface for microbubbles comprising such structures.
The lipopeptides described above form a further aspect of the invention and may be natural, semisynthetic or synthetic in origin, although the lipopeptides of the invention are preferably produced synthetically. Thus, the invention also provides a membrane-forming amphiphilic peptide of general formula:
Axe2x80x94B
(wherein A represents a peptide comprising from 2 to 50 residues and B represents one or more hydrocarbon chains of between 5 and about 50 carbons).
In one embodiment, one or more of the peptide termini or available side-chains may be coupled to a polyethylene glycol derivative in order to delay uptake by the reticulo-endothelial system. Polyethylene glycol derivatives are also considered useful in reducing opsonisation of the microbubbles by serum proteins. This is considered especially relevant when targeting of the microbubbles is desirable.
In a further embodiment, multifunctional aromatic systems may be used to link the peptides and lipophilic moieties of the invention to enhance membrane stability. The presence of aromatic systems may further strengthen intermolecular associations within the membrane due to Π-Π stacking interactions. The aromatic group, which may be a carbocyclic or heterocyclic, mono- or polycylic aryl group, is advantageously phenyl. It may link one or more peptides along with one or more hydrophobic hydrocarbon residues. Conveniently, the peptide(s) may be linked to the aromatic system via an amide linkage; for example the N-terminus of a suitable peptide may be coupled to a benzoic acid derivative. One or more hydrophobic groups such as fatty acid derivatives may be linked directly to the aromatic group, for example via an amide linkage, or may be connected to the aromatic group by a suitable linker or linkers. In a preferred embodiment such lipopeptides may be represented by the formula: 
where A is an alkyl chain linked to the phenyl ring by a suitable linker, e.g. an amide group, B is either an alkyl chain linked to the phenyl ring by a suitable linker or a peptide sequence as hereinbefore described linked to the phenyl ring by a suitable linker and C is a peptide sequence as hereinbefore described linked to the phenyl ring by a suitable linker.
Preferably the substituents should be at the 1,3 and 5 positions of the phenyl ring.
A particularly preferred aromatic system is based on 3,5-diaminobenzoic acid. The diaminobenzoic acid scaffold allows for differential coupling without complicated protection strategies being employed. This is due to the reduced reactivity of the second amino group following acylation of the first amino group.
Suitable linking groups for attachment of a hydrocarbon chain or peptide to the aromatic system include amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl groups, imidazolyl groups, phenolic groups and xcex1-haloacetyl compounds of the type Xxe2x80x94CH2COxe2x80x94 (where X=Br, Cl or I). Other linking moieties will of course be readily determined by the person skilled in the art.
The aromatic linked lipopeptides described above form a further aspect of the invention.
In order to form an encapsulating membrane, a homogeneous preparation of a single lipopeptide component or heterogeneous mixtures of two or more complementary lipopeptide components may be used. Preferably a mixture of two complementary lipopeptide components is employed.
The membranes of the microbubbles of the invention may comprise one or more mono-, di- or multi-valent metal ions and, although not wishing to be limited by theory, it is believed that the metal ions may play a role in the stabilisation of the membrane. Suitable metal ions include gadolinium (III), yttrium (III) and calcium (II), but preferably the metal ion will be monovalent, e.g a sodium or potassium ion. The presence of metal ions in the membrane may also facilitate compatibilty with buffering systems and may confer some complexing or chelating stability on the membrane.
In a further embodiment of the invention gas-filled lipopeptide microbubbles incorporating chelates which bind metal ions such as gadolinium, indium or technecium may be prepared. Lipopeptides suitable for iodination, e.g. tyrosine-containing lipopeptides, may form the encapsulating membrane. In this way multi-modality imaging may be carried out.
The microbubble membrane may be a monolayer, bilayer, oligolamellar or a fibrous network of interwoven lipopeptides, for example depending on the method of preparation.
Any biocompatible gas may be present in the microbubbles according to the invention, the term xe2x80x9cgasxe2x80x9d as used herein including any substances (including mixtures) substantially or completely in gaseous (including vapour) form at the normal human body temperature of 37EC. The gas may thus, for example, comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon or krypton; a sulphur fluoride such as sulphur hexafluoride, disulphur decafluoride or trifluoromethylsulphur pentafluoride; selenium hexafluoride; an optionally halogenated silane such as methylsilane or dimethylsilane; a low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms), for example an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne; an ether such as dimethyl ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing. Advantageously at least some of the halogen atoms in halogenated gases are fluorine atoms; thus biocompatible halogenated hydrocarbon gases may, for example, be selected from bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethylfluoride, 1,1-difluoroethane and perfluorocarbons, e.g. perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-iso-butane), perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2-ene) and perfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (e.g. perfluorinated) ketones such as perfluoroacetone and fluorinated (e.g. perfluorinated) ethers such as perfluorodiethyl ether. The use of perfluorinated gases, for example sulphur hexafluoride and perfluorocarbons such as perfluoropropane, perfluorobutanes and perfluoropentanes, may be particularly advantageous in view of the recognised high stability in the bloodstream of microbubbles containing such gases.
Gas microbubbles preferably have an initial average size not exceeding 10 xcexcm (e.g. of 7 xcexcm or less) in order to permit their free passage through the pulmonary system following administration, e.g. by intravenous injection. However, larger microbubbles may be employed where, for example, these contain a mixture of one or more relatively blood-soluble or otherwise diffusible gases such as air, oxygen, nitrogen or carbon dioxide with one or more substantially insoluble and non-diffusible gases such as perfluorocarbons. Outward diffusion of the soluble/diffusible gas content following administration will cause such microbubbles rapidly to shrink to a size which will be determined by the amount of insoluble/non-diffusible gas present and which may be selected to permit passage of the resulting microbubbles through the lung capillaries of the pulmonary system.
The lipopeptide structures discussed above may advantageously enhance membrane stability by allowing for intermolecular association through a combination hydrophobic, ion-pairing and hydrogen bonding interactions. Hydrogen bonding may occur between donor and acceptor atoms on juxstaposed lipopeptide chains. Hydrophobic interactions may occur between hydrophobic moieties such as alkyl chains or a sequence of hydrophobic amino acid residues, so as to form a hydrophobic inner core of the membrane.
One preferred aspect of this invention relates to the targeting of ultrasound microbubbles for disease imaging and drug delivery. Thus, viewed from another aspect the invention provides a targeted diagnostic and/or therapeutically active agent, e.g. an ultrasound contrast agent, comprising (i) gas-filled microbubbles stabilised by membrane forming amphiphilic lipopeptides capable of interacting with ultrasound irradiation to generate a detectable signal; (ii) one or more vector or drug molecules or a combination of both, where said vector(s) have affinity for a particular target site and/or structures within the body, e.g. for specific cells or areas of pathology; and (iii) one or more linkers connecting said microbubbles and vectors, in the event that these are not directly joined.
The use of vectors to target specific areas of interest within the body is well-known in the art and their use will be routine to the skilled artisan. Suitable vectors of use in the present invention include protein and peptide vectors such as antibodies, cell adhesion molecules such as L-selectin, RGD-peptides, PECAM and intergrin, vectors comprising cytokines/growth factors/peptide hormones and fragments thereof, streptavidin, bacterial fibronectin-binding proteins, Fc-part of antibodies, transferrin, streptokinase/tissue plasminogen activator, plasminogen, plasmin, mast cell proteinases, elastase, lipoprotein, lipase, coagulation enzymes, extracellular superoxide dismutase, heparin cofactor II, retinal survival factor, heparin-binding brain mitogen, apolipoprotein (e.g. apolipoprotein B or apolipoprotein E), adhesion-promoting proteins (e.g. purpurin), viral coat proteins (e.g. from HIV or herpes), microbial adhesins (e.g. xcex2-amyloid precursor), tenascin (e.g. tenascin C), vectors comprising non-peptide agonists/antagonists of cytokines/growth factors/peptide hormones/cell adhesion molecules, vectors comprising anti-angiogenic factors, vectors comprising angiogenic factors, vector molecules other than recognized angiogenetic factors which have known affinity for receptors associated with angiogenesis, receptors/targets associated with angiogenesis, oligonucleotide vectors, modified oligonucleotide vectors, nucleoside and nucleotide vectors, receptors comprising DNA-binding drugs, receptors comprising protease substrates, receptors comprising protease inhibitors, vectors from combinatorial libraries, carbohydrate vectors, lipid vectors and small molecule vectors such as adrenalin and betablockers.
The microbubbles of the invention may be coupled to one or more vectors either directly or through linking groups. The microbubbles may be coupled to vectors such as monoclonal antibodies which recognise specific target areas or to a secondary antibody which has a specificity for a primary antibody which in turn has specificity for a target area. Such use of secondary antibodies is advantageous in that appropriate selection of a secondary antibody allows the preparation of xe2x80x9cuniversalxe2x80x9d microbubbles which may be used for a wide range of applications, since the primary antibody can be tailored to particular target areas.
Coupling of a microbubble to a desired vector may be achieved by covalent or non-covalent means for example involving interaction with one or more functional groups located on the microbubble and/or vector. Examples of chemically reactive groups which may be employed for this purpose include amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl groups, imidazolyl groups and phenolic groups. The vector and microbubble may also be linked by a linking group; many such groups are well-known in the art. Connection of the linker to the vector and microbubble may be achieved using routine synthetic chemical techniques. A comprehensive summary of known vectors and linking groups useful in targeting ultrasonic echography can be found in International Patent Publication No. WO-A-9818501, the contents of which are hereby incorporated by reference.
The present invention also provides a tool for therapeutic drug delivery in combination with vector-mediated direction of the product to the desired site. By xe2x80x9ctherapeutic drugxe2x80x9d is meant an agent having a beneficial effect on a specific disease in a living human or non-human animal. Whilst combinations of drugs and ultrasound contrast agents have been proposed in, for example, WO-A-9428873 and WO-A-9507072, these products lack vectors having affinity for particular sites and thereby show comparatively poor specific retention at desired sites prior to or during drug release.
Therapeutic compounds used in accordance with the present invention may be encapsulated in the interior of the microbubbles or attached to or incorporated in the encapsulating walls. Thus, the therapeutic compound may be linked to a part of the wall, for example through covalent or ionic bonds, or may be physically mixed into the encapsulating material, particularly if the drug has similar polarity or solubility to the membrane material, so as to prevent it from leaking out of the product before its intended action in the body. Release of the drug may be initiated merely by wetting contact with blood following administration or as a consequence of other internal or external influences, e.g. dissolution processes catalyzed by enzymes or the use of of ultrasound. The destruction of gas-containing microparticles using external ultrasound is a well known phenomenon in respect of ultrasound contrast agents, e.g. as described in WO-A-9325241; the rate of release may be varied depending on the type of therapeutic application by using a specific amount of ultrasound energy from the transducer.
The therapeutic agent may be covalently linked to is the encapsulating membrane surface using a suitable linking agent. Thus, for example, one may initially prepare a lipopeptide derivative to which the drug is bonded through a biodegradable or selectively cleavable linker, followed by incorporation of the material into the microbubble. Alternatively, lipidated drug molecules which do not require processing to liberate an active drug may be incorporated directly into the membrane. The active lipidated drug may, for example, be released by increasing the strength of the ultrasound beam.
Exemplary drug delivery systems suitable for use in the present compositions include known therapeutic drugs or active analogues thereof containing thiol groups; these may be coupled to thiol group-containing microbubbles under oxidative conditions yielding disulphide bridges. In combination with a vector or vectors such drug/vector modified microbubbles may be allowed to accumulate in the target tissue; administration of a reducing agent such as reduced glutathione will then liberate drug molecules from the targeted microbubbles in the vicinity of the target tissue, increasing the local concentration of the drug and enhancing its therapeutic effect. It is also possible to prepare microbubbles which may be coupled to or coated with a therapeutic drug immediately prior to use. Thus, for example, a therapeutic drug may be added to a suspension of such microbubbles in an aqueous medium and shaken in order to attach or adhere the drug to the microbubbles.
A comprehensive summary of the use of microbubbles in drug delivery applications can be found in the aforementioned WO-A-9818501.
The lipopeptides of the invention may, for example, be prepared by conventional peptide synthesis techniques using appropriate protection. The synthesis may conveniently be carried out using an automatic peptide synthesiser, for example using the Merrifleld solid phase peptide synthesis technique. Hydrocarbon chains may be coupled to the peptide at any convenient stage, e.g. before a residue has been incorporated into a peptide or after the entire peptide has been synthesised, for example using standard organic chemistry procedures. It is preferred that any hydrocarbon chain carries a carboxylate functionality such as an acyl chloride moiety or carboxylic acid group which may readily be coupled onto a free amino side chain or the N-terminus of the peptide. If the peptide and lipophilic components are to be linked via a aromatic system such as 3,5-diaminobenzoic acid, binding to the aromatic system will be readily effected by the skilled artisan. For example, a peptide may be coupled to the carboxyl acid group of 3,5-diaminobenzoic acid by simple peptide synthesis. A fatty acid may then be coupled to one amino functional groups to yield a 1,3-disubstituted derivative; such reaction with one amino group deactivates the other free amino functionality, so that a 1,3,5-trisubstituted compound does not result. The 1,3-disubstituted derivative may then be coupled further with a desired peptide or lipophilic group, again using simple synthetic chemistry procedures, but using more severe reaction conditions.
Microbubbles according to the invention may, for example, be prepared by sonicating and warming an aqueous solution comprising the required lipopeptide(s) and optionally also any metal ions and/or other desired components, while exposing the solution to an appropriate gas. Other techniques for the preparation of microbubbles, as well as appropriate isolation and purification procedures, are well known in the art.